Asthma is an inflammatory disease of lung airways that makes the airways prone to narrow too much and too easily in response to a wide variety of provoking stimuli. In the lung, the major innervating sensory and motor nervous system is contained within the vagus nerve (FIG. 1). Exposure of the airway to irritants such as sulfur dioxide, prostaglandins, histamine and cold air can stimulate afferent sensory fibers of the vagus nerve, thereby setting off bronchoconstriction, or airway narrowing, due to reflex release of acetylcholine by cholinergic efferent motor branches of the vagus nerve. While this reflex is present in normal individuals, it is greatly exaggerated in asthmatic patients. This exaggerated narrowing is often called airways hyperreactivity.
Airways hyperreactivity in asthmatic patients and in animal models of asthma is thought to arise from increased release of the endogenous neurotransmitter acetylcholine from the efferent motor vagus nerve endings innervating the airway (A. D. Fryer, et al., Journal of Clinical Investigation (1992) 90:2292-2298). In the airway, release of acetylcholine from the vagus nerves is under the local control of inhibitory muscarinic autoreceptors on the postganglionic nerves (FIG. 1). These autoreceptors are called M.sub.2 muscarinic receptors, while the muscarinic receptors on airway smooth muscle are M.sub.3 receptors. Thus, acetylcholine released from the vagus nerve stimulates both M.sub.3 muscarinic receptors on airway smooth muscle, causing bronchoconstriction, and M.sub.2 muscarinic receptors on the nerves, decreasing further release of acetylcholine. In asthmatics, inhibitory M.sub.2 muscarinic receptors are dysfunctional, resulting in exaggerated acetylcholine release and, therefore, exaggerated bronchoconstriction, or airways hyperreactivity, in response to a given irritant airway stimulus (A. D. Fryer, el al., Journal of Clinical Investigation (1992) 90:2292-2298; D. B. Jacoby, el al., Journal of Clinical Investigation (1993) 91:1314-1318).
The negative feedback control of acetylcholine release provided by the M.sub.2 muscarinic receptor can be demonstrated experimentally by measuring vagally induced bronchoconstriction in the presence of selective muscarinic agonists or antagonists. Blockade of neuronal muscarinic M.sub.2 receptors with gallamine potentiates vagally induced bronchoconstriction. Conversely, the selective muscarinic M.sub.2 receptor antagonist pilocarpine inhibits irritant-induced cholinergic reflex bronchoconstriction in normal subjects. This inhibitory mechanism is not present in asthmatics because of dysfunctional M.sub.2 receptors (P. A. Minette, et al., Journal of Applied Physiology (1989) 67:2461-2465). Such a defect in muscarinic autoreceptors results in exaggerated cholinergic reflexes in asthma, because the normal feedback inhibition of acetylcholine release is lost.
M.sub.2 receptor dysfunction and subsequent airways hyperreactivity in asthma is thought to be due to increased susceptibility of the receptor to damage by products of the inflammatory response in the airway. Asthma results in an influx of inflammatory cells, especially eosinophils, into the airway. Activated eosinophils in asthmatics secrete a number of injurious proteins, including major basic protein, eosinophil peroxidase, and eosinophil cationic protein. All of these proteins are strongly positively charged. These and other positively charged proteins can cause airway hyperresponsiveness (R. H. Gundel, et al., Journal of Clinical Investigation (1991) 87:1470-1473; A. J. Coyle, et al., American Review of Respiratory Diseases (1993) 147:896-900). Major basic protein (D. B. Jacoby, et al., Journal of Clinical Investigation (1993) 91:1314-1318) and other positively charged proteins (J. Hu, el al. Molecular Pharmacology (1992) 42:311-324) have been shown to function as M.sub.2 muscarinic receptor antagonists. Thus, airways hyperreactivity in asthma is a consequence of direct antagonism of inhibitory M.sub.2 cholinergic receptors by components of airway inflammation.
The treatment of airways hyperreactivity in asthma is currently directed against either inhibiting the airway inflammation leading to release of products that inhibit M.sub.2 receptors, or toward direct reversal of bronchoconstriction of airway smooth muscle. Corticosteroids are the mainstay of anti-inflammatory therapy. Beta-adrenergic agonists, acting by stimulation of beta.sub.2 adrenergic receptors on airway smooth muscle, are used as bronchodilators to directly reverse constricted airways. Nonselective anti-cholinergic drugs such as atropine and ipratropium bromide are available for use as bronchodilators, but block both prejunctional M.sub.2 receptors and M.sub.3 receptors on smooth muscle with equal efficacy. This increases acetylcholine release, overcoming the postjunctional blockade, and makes these nonselective anti-cholinergic agents ineffective at reversing vagally mediated bronchoconstriction. A more specific treatment for reversing the M.sub.2 receptor blockade would be of great benefit as a treatment for the airways hyperreactivity of asthma.
Recently, the anticoagulant drug heparin has been shown to reverse antigen-induced M.sub.2 receptor dysfunction in antigen-challenged guinea pigs (A. D. Fryer, et al., Journal of Clinical Investigation (1992) 90:2292-2298) and to reverse binding of M.sub.2 receptor by major basic protein it vitro (D. B. Jacoby, el al., Journal of Clinical Investigation (1993) 91:1314-1318). Heparin has over the years been suggested as a treatment for asthma (M. M. Hartman, California Medicine (1 963) 98:27-32; D. A. Dolowitz, et al., Annals of Allergy (1 965) 23:309-313; T. Ahmed, el al., American Review of Respiratory Diseases (1992) 145:566-570; T. Ahmed, el al., Journal of Applied Physiology (1993) 74:1492-1498; S. D. Bowler, et al., American Review of Respiratory Diseases (1993) 147:160-163; T. Ahmed, el al., New England Journal of Medicine; International PCT Application, PCT/US93/02880). However, as a treatment for the airways hyperreactivity of asthma, heparin has one great disadvantage: it is an anticoagulant. As such, it would expose the treated patient to an unacceptable risk of hemorrhage, even if treatment was localized by aerosolization of heparin into the lung airway. Aerosolized heparin is well absorbed into the systemic circulation, and administration of heparin by lung aerosolization has been advocated as a method of anticoagulating the blood (L. B. Jaques, el al., Lancet (1976) ii:157-1161).
To use heparin safely as a treatment for the airways hyperreactivity of asthma, it would need to be first inactivated as an anticoagulant without affecting its efficacy to treat asthma. Several chemical methods exist for inactivating heparin as an anticoagulant. Most are based on techniques of chemical desulfation, since it is well established that sulfate groups of heparin are important for anticoagulant activity. However, N-desulfated heparin has been previously reported to be ineffective in the prevention of asthmatic-like bronchoconstriction from aerosolized antigen (T. Ahmed, et al., American Review of Respiratory Diseases (1992) 145:566-570, see FIG. 2). Additionally, N-desulfated heparin has been previously reported to be only 50% as effective as heparin in complement inhibition (J. M. Weiler et al., J.Immunol. (1992) 148:3210-3215; R. E. Edens et al. Complement Today (Cruse, J. M. and Lewis, R. E. Jr. eds): Complement Profiles (1993) 1:96-120).
Thus, the literature teaches that chemical desulfation would not be an effective strategy in modifying heparin for use as an effective treatment for asthmatic airways hyperreactivity. In contrast to what would be predicted by the literature, the present invention discloses that, surprisingly, selective O-desulfation of heparin eliminates the anticoagulant activity of heparin without destroying the ability of heparin to reverse M.sub.2 muscarinic receptor blockade in asthma.
Asthma has been long described in the medical literature as an episodic disease characterized by reversible airways obstruction. This is in contrast to chronic obstructive airways disease from chronic bronchitis and emphysema, in which physiologic airways obstruction is permanent and slowly progressive. However, the characterization of airways obstruction in asthma as episodic and reversible may be simplistic. Clinical pulmonary physicians have recently begun to appreciate a population of asthmatics, usually older individuals, who appear to have unrelenting disease, with lung function that never normalizes between acute bronchospastic episodes. Some of these patients appear to progress to fixed airways obstruction without the presence of other known risk factors such as active or past cigarette smoking. This population presents a difficult clinical challenge, in that many of these individuals are steroid dependent or even relatively resistant to intervention with steroids and other anti-inflammatory or bronchodilator medications.
One possible explanation for this difficult-to-treat population is that patients with chronic asthma undergo remodeling of their airways, with substantial increase in the amount of smooth muscle in airway walls (Heard, B. E., and S. Hossain. 1973. Hyperplasia of bronchial muscle in asthma. J. Path. 110:319-331, James, A. L., P. D. Pare, and J. C. Hogg. 1989. The mechanics of airway narrowing in asthma. Am. Rev. Respir. Dis. 139:242-246; Saetta, M., A. DiStefano, C. Rosina, G. Thiene, and L. M. Fabbri. 1991. Quantitative structural analysis of peripheral airways and arteries in sudden fatal asthma. Am. Rev. Respir. Dis. 143:138-143; Ollerenshaw, S. L., and A. J. Woolcock. 1992. Characteristics of the inflammation in biopsies from large airways of subjects with asthma and subjects with chronic airflow limitation. Am. Rev. Respir. Dis. 145:922-927). Patients dying of asthma have over twice the amount of airway smooth muscle as nonasthmatic subjects (Saetta, M., A. DiStefano, C. Rosina, G. Thiene, and L. M. Fabbri. 1991. Quantitative structural analysis of peripheral airways and arteries in sudden fatal asthma. Am. Rev. Respir. Dis. 143:138-143), and airway smooth muscle hypertrophy is seen in sensitized Brown-Norway rats (Sapienza, S., T. Du, D. H. Eidelman, N. S. Wang, and J. G. Martin. 1991. Structural changes in the airways of sensitized Brown Norway rats after antigen challenge. Am. Rev. Respir. Dis. 144:423-427; Wang, C. G., T. Du, L. J. Xu, and J. G. Martin. 1993. Role of leukotriene D.sub.4 in allergen-induced increases in airway smooth muscle in the rat. Am. Rev. Respir. Dis. 148:413-417) and cats (Padrid, P., S. Snook, T. Finucane, P. Shiue, P. Cozzi, J. Solway, and A. R. Leff. 1995. Persistent airway hyperresponsiveness and histologic alterations after chronic antigen challenge in cats. Am. J. Respir. Crit. Care Med. 151:184-193) after antigen challenge. Increased airway smooth muscle might be expected to change the counterbalance of forces tending to distend or close the airway lumen, thereby altering the location of the equal pressure point, when air is unable to flow (Pride, N. B., S. Permutt, R. L. Riley, and B. Bromberger-Barnea. 1967. Determinants of maximal expiratory flow from the lungs. J. Appl. Phlysiol 23:646-662). Airway wall thickening has also been proposed as a partial explanation for exaggerated changes in airway caliber when airway smooth muscle shortens (James, A. L., P. D. Pare, and J. C. Hogg. 1989. The mechanics of airway narrowing in asthma. Am. Rev. Respir. Dis. 139:242-246). Even small changes in airway wall thickness that have little effect on baseline resistance to airflow can produce an increase in maximal airway responsiveness to agonists, similar to that seen in asthmatics (Moreno, R. H., J. C. Hogg, and P. D. Pare. 1985. Mechanisms of airway narrowing. Am. Rev. Respir. Dis. 133:1171-1180).
The precise stimuli for airway smooth muscle hypertrophy in asthma are unclear, but several possible mitogens for airway smooth muscle have been demonstrated, including endothelin, histamine, the mast cell enzyme tryptase and leukotrienes (Wang, C. G., T. Du, L. J. Xu, and J. G. Martin. 1993. Role of leukotriene D.sub.4 in allergen-induced increases in airway smooth muscle in the rat. Am. Rev. Respir. Dis. 148:413-417; Vitori, E. N., M. Marini, A. Fasoli, R. De Franchia, and S. Mattoli. 1992. Increased expression of endothelin in bronchial epithelial cells of asthmatic patients and effect of corticosteroids. Am. Rev. Respir. Dis. 146:1320-1325; Noveral, J. P., S. M. Rosenberg, R. A. Anbar, N. A. Pawlowski, and M. M. Grunstein. 1992. Role of endothelin-1 in regulating proliferation of cultured rabbit airway smooth muscle cells. Am. J. Physiol. 263(Lung Cell. Mol Physiol. 7):L317-L324; Glassberg, M. K., A. Ergul, A. Wanner, and D. Puett. 1994. Endothelin-1 promotes mitogenesis in airway smooth muscle cells. Am. J. Respir. Cell Mol. Biol. 10:316-321; Panettieri, R. A., P. A. Yadvish, A. M. Kelly, N. A. Rubinstein, and M. I. Kotlikoff. 1990. Histamine stimulates proliferation of airway smooth muscle and induces c-fos expression. Am. J. Physiol 259 (Lung. Cell Mol. Physiol. 3):L365-L371; Ruoss, S. J., T. Hartmann, and G. Caughey. 1992. Mast cell tryptase is a mitogen for cultured fibroblasts. J Clin. Invest. 88:493-499) The polycation protamine is mitogenic for cultured vascular smooth muscle (Edelman, E. R., L. A. Pukac, and M. J. Karnovsky. 1993. Protamine and protamine-insulins exacerbate the vascular response to injury. J Clin. Invest. 91:2308-2313). Therefore, it is also possible that eosinophil-derived positively-charged polycations such as major basic protein might stimulate proliferation of airway smooth muscle.
Equally unclear is how airway smooth muscle remodeling in asthma might be prevented. The bronchodilator salbutamol inhibits proliferation of cultured human airway smooth muscle in response to thrombin and epidermal growth factor (Tomlinson, P. R., J. W. Wilson, and A. G. Stewart. 1994. Inhibition by salbutamol of the proliferation of human airway smooth muscle cells grown in culture. Br. J. Pharmacol. 111:641-647). However, in general, by preventing mast cell degranulation, beta adrenergic agonist bronchodilators may deprive the airway of the anti-proliferative effects of mast cell heparin release, thereby exacerbating smooth muscle remodeling (Page, C.P. 1991. One explanation of the asthma paradox: inhibition of natural anti-inflammatory mechanism by B.sub.2 -agonists. Lancet 337:717-720). In the chronically antigen challenged ovalbumin-sensitized Brown Norway rat, the leukotriene D.sub.4 antagonist MK-571 reduces smooth muscle proliferation of small airways, but was only partially effective in preventing airway remodeling of larger airways (Wang, C. G., T. Du, L. J. Xu, and J. G. Martin. 1993. Role of leukotriene D.sub.4 in allergen-induced increases in airway smooth muscle in the rat. Am. Rev. Respir. Dis. 148:413-417). Because more than one mitogen is likely to promote smooth muscle proliferation in asthmatic patients, it is not surprising that specific blockade of one mediator fails to prevent the remodeling process. For therapy, a treatment is needed that intervenes at a more focal control point in growth regulatory events.
Mast cell heparin has been proposed to normally modulate growth and proliferation of airway smooth muscle (Page, C.P. 1991. One explanation of the asthma paradox: inhibition of natural anti-inflammatory mechanism by B.sub.2 -agonists. Lancet 337:717-720). The closely related sulfated polysaccharide heparan sulfate has been shown to inhibit proliferation of cultured canine tracheal smooth muscle (Panettieri, R. A., P. A. Yadvish, A. M. Kelly, N. A. Rubinstein, and M. I. Kotlikoff. 1990. Histamine stimulates proliferation of airway smooth muscle and induces c-fos expression. Am. J. Physiol. 259 (Lung Cell. Mol. Physiol. 3):L365-L371). Heparin is a potent inhibitor of proliferation of vascular smooth muscle in vitro (Hoover, R. L., R. Rosenberg, W. Haering, and M. J. Karnovsky. 1980. Inhibition of rat arterial smooth muscle cell proliferation by heparin. Cir. Res. 47:578-583) and in vivo (Guyton, J. R., R. D. Rosenberg, A. W. Clowes, and Karnovsky. 1980. Inhibition of rat arterial smooth muscle cell proliferation by heparin. In vivo studies with anticoagulant and nonanticoagulant heparin. Cir. Res. 46:625-634; Clowes, A. W., and M. M. Clowes. 1985. Kinetics of cellular proliferation after arterial injury. II. Inhibition of smooth muscle growth by heparin. Lab. Invest. 42:611-616; Clowes, A. W., and M. M. Clowes. 1986. Kinetics of cellular proliferation after arterial injury. IV. Heparin inhibits rat smooth muscle mitogenesis and migration. Circ. Res. 58:839-845).
Recently, heparin and low molecular weight heparin have been demonstrated by Kilfeather el al. to be potent inhibitors of serum-induced proliferation of bovine tracheal smooth muscle cells in culture (Kilfeather, S. A., S. Tagoe, A. C. Perez, K. Okona-Mensa, R. Matin, and C. P. Page. 1995. Inhibition of serum-induced proliferation of bovine tracheal smooth muscle cells in culture by heparin and related glycosaminoglycans. Brit. J. Pharamcol. 114:1442-1446). In discussing structure-activity implications of their findings, Kilfeather and coworkers suggested that O-sulfation is required for antiproliferative activity in airway smooth muscle cells. Earlier, Wright et al, had shown that increasing the charge of inactive tetrasaccharide fragments by O-oversulfation made them antiproliferative against vascular smooth muscle, whereas reducing the charge of active larger fragments caused them to lose their antiproliferative activity (Wright, T. C., Jr., J. J. Castello, Jr., M. Petitou, J.-C. Lormeau, J. Choay, and M. J. Karnovsky. 1989. Structural determinants of heparin's growth inhibitory activity. Interdependence of oligosaccharide size and charge. J Biol. Chem. 264:1534-1542). Castellot et al, had suggested an absolute requirement for 3-O sulfation as a necessary structural requirement for heparin to inhibit vascular smooth muscle proliferation (Castellot, J. J., Jr., J. Choay, J.-C. Lormeau, M. Petitou, E. Sache, and M. J. Karnovsky. 1986. Structural determinants of the capacity of heparin to inhibit the proliferation of vascular smooth muscle cells. II. Evidence for a pentasaccharide sequence that contains a 3-O-sulfate group. J. Cell Biol. 102:1979-1984). Maccarana et al. reported the importance of 2-O sulfates for heparin binding of the mitogen basic fibroblast growth factor (Maccarana, M., B. Casu, and U. Lindahl. 1993. Minimal sequence in heparin/heparan sulfate required for binding of basic fibroblast growth factor. J. Biol. Chem. 268:23898-23905).
In contrast, the present invention provides the surprising discovery that a selectively 2-O, 3-O-desulfated heparin produced by alkaline lyophilization is a potent inhibitor of fetal calf serum stimulated-airway smooth muscle proliferation.
2-O-desulfated heparin has been reported to be made (R. Rej el al., Thrombosis and Hemostasis (1989)61:540; and M. Jaseja et al., Canadian Journal of Chemistry (1989) 67:1449-1456). Actually, those authors did not recognize that the compound they made was, in fact, 2-O as well as 3-O desulfated heparin. Briefly, the Rej et al. and Jaseja et al. method comprises starting with a heparin solution pH adjusted with 0.1 N sodium hydroxide, which is then lyophilized to produce a 2-O-desulfated alpha-L-iduronic acid residue (and a 3-O-desulfated glucosamine residue). The anticoagulant activity of heparin was studied; however, there was no suggestion of inhibition of airways reactivity or treatment of asthmatic conditions. Likewise, Rej el al. and Jaseja et al. disclosed no activity for 2-O, 3-O-desulfated heparin, and further, did not disclose any effective doses for the compound for any purpose.